Evaluation of adenovirus vectors containing serotype 35 fibers for tumor targeting.

There is growing evidence from in vitro studies that subgroup B adenoviruses (Ad) can overcome the limitations in safety and tumor transduction efficiency seen with commonly used subgroup C serotype 5-based vectors. In this study, we confirm that the expression level of the B-group Ad receptor, CD46, correlates with the grade of malignancy of cervical cancer in situ. We also demonstrate the in vivo properties of Ad5-based vectors that contain the B-group Ad serotype 35 fiber (Ad5/35) in transgenic mice that express CD46 in a pattern and at a level similar to humans. Upon intravenous and intraperitoneal injection, an Ad5/35 vector did not efficiently transduce normal tissue, but was able to target metastatic or intraperitoneal tumors that express CD46 at levels comparable to human tumors. When an oncolytic Ad5/35-based vector was employed, in both tumor models antitumor effects were observed. Furthermore, injection of Ad5/35 vectors into CD46 transgenic mice caused less innate toxicity than Ad5 vectors. Our data demonstrate that Ad vectors that target CD46 offer advantages over Ad5-based vectors for treatment of cancer.

Genetic variability and function of the long terminal repeat from syncytium-inducing and non-syncytium-inducing human immunodeficiency virus type 1.

We analyzed sequence variability and function of the long terminal repeat (LTR) from syncytium-inducing (SI) and non-syncytium-inducing (NSI) HIV-1. Twenty LTR DNA clones were obtained by polymerase chain reaction amplification and molecular cloning from short-term cultures of SI and NSI viruses from an AIDS patient and two asymptomatic individuals, respectively. All the LTR clones tested contained multiple nucleotide changes (mostly G-to-A transitions), compared to the subtype B consensus sequence, which were clustered within the negative regulatory element, including NF-AT, USF, and TCF-1 alpha binding sites. The core promoter/TAR region sequences were highly conserved. The basal and Tat-mediated transcriptional activities of selected LTR clones tested were 0.1 to 1 and 0.2 to 0.5 times that of the control, respectively, regardless of the SI or NSI origin of the clones. Phylogenetic analysis revealed interi-solate sequence divergence in the LTR that was similar but not identical to previously analyzed vif sequences from the same samples. In particular, the inter-isolate distances from reference sequences differed for the LTR and vif. This raises the possibility that recombination occurred between corresponding LTR and vif loci of the quasi-species present in the isolates described here.

Bioequivalence of two rimantadine tablet formulations in healthy male volunteers after single dose administration.

AIM: The bioequivalence of two rimantadine tablet formulations was determined. METHODS: The study was designed as a randomized, two-period, two-sequence, crossover study. Twenty-four healthy male volunteers received a single 100 mg dose of rimantadine hydrochloride as test (Rimantadin Lachema 100 tbl. obd., produced by Lachema, a.s., Brno, Czech Republic) and reference formulations (Elumadine 100 tbl. obd., produced by Forest Pharmaceuticals, St. Louis, USA). The two administrations were separated by 14 days and were performed in the fasting state. Blood samples were obtained at 15 time points during the interval 0-120 h after administration. Rimantadine plasma concentrations were determined by gas chromatography with electron-capture detection. RESULTS: The geometric mean concentration-time profiles of rimantadine after administration of the two formulations were superimposable. The following pharmacokinetic parameters refer to the geometric mean [exp(mean +/- SD)] values for the test and reference formulations, respectively: Cmax (ng/ml) 70.5 (60.0-82.7) vs. 70.0 (59.9 to 81.7), AUC(0-infinity) (ng x h/ml) 2872 (2224 to 3707) vs. 2849 (2195-3699), AUC(0-120 h) 2744 (2184-3448) vs. 2712 (2138-3441), t(1/2) (h) 25.8 (20.1-33.0) vs. 25.7 (20.6 to 32.1). Median (range) tmax (h) values were 4.5 (2.0-8.0) and 6.0 (2.0-8.0). Parametric 90% confidence intervals for the expected mean percentage ratios (test/reference) of the pharmacokinetic variables were within the range of 97% to 105%. The median (91.1% confidence interval) difference in tmax was -0.3 h (-2.0-0.5). The point and interval estimates were identical when truncated AUCs (0-96 h, 0-72 h, 0-48 h and 0-24 h) were used in calculations. CONCLUSION: The two rimantadine formulations were equivalent in both the rate and extent ofbioavailability and they were also well tolerated. This study confirms the findings of other studies showing that for immediate release formulations of drugs with long half-lives shortening the duration over which blood samples are collected improves the economics, is more ethical and does not impair the quality of data.

Antibody against synthetic peptide derived from Epstein-Barr virus-determined nuclear antigen 1 (EBNA-1) in child non-Hodgkin's lymphoma.

Antibody reactivity against a synthetic peptide derived from Epstein-Barr virus nuclear antigen 1 (EBNA-1) was determined in 56 cases of child non-Hodgkin's lymphoma and 31 controls. The patients were divided into subgroups based on tumour location and histology and the antibody responses in the various groups were compared. A significant increase in both IgG and IgM antipeptide titres was detected in patients with tumours localized in the abdomen. High IgG titres were also noted in Burkitt-type, lymphoblastic, and centroblastic lymphomas. On the other hand, low or nil IgG titres were found in unclassified malignant lymphomas, in four cases of centroblastic-centrocytic lymphoma and in lymphomas located in the mediastinum. Surprisingly, the occurrence of antipeptide IgM antibody was highest in those tumours, where IgG titres were low, i.e. in subjects with mediastinal tumours and in unclassified malignant lymphomas. However, with the exception of tumours localized in the abdomen and unclassified tumours, the IgM titres in positive individuals were low and comparable with titres found in a part of healthy controls.

[Pharmaceutical significance of Allium sativum L. 4. Antifungal effects]. / Farmaceutický význam Allium sativum L. 4. Antifungální úcinek.

The recent decades saw an increase in the number of systemic fungal diseases and improvements in the identification of their causative agents. There has been an intensive search for new drugs which would be more effective and less toxic than those already in use. From this aspect, attention has been paid also to garlic--its extracts and individual components, i.e., allicin, ajoen, polysulfides, essential oil. New experimental knowledge confirms a significant antifungal activity of sulfurous compounds of garlic. The paper also mentions a possible use of employing garlic extracts or essential oils in food industry as an alternative way of protection of foodstuffs from contamination with fungi.

[Pharmaceutical importance of Allium sativum L. 5. Hypolipemic effects in vitro and in vivo]. / Farmaceutický význam Allium sativum L. 5. Hypolipidemický úcinek in vitro a in vivo.

The paper summarizes the knowledge on the antihyperlipidemic effect of garlic, formulations prepared from it, and the individual components, which were obtained prevalently in recent decade. It presents varying opinions based on experimental results concerning the mechanisms by means of which the effect takes place. In vitro experiments were carried out mainly on the cultures of rat hepatocytes and an inhibitory effect on important enzymic activities taking place in the biosynthesis of cholesterol and fatty acids was demonstrated. The most frequently employed in vivo models were rabbits. The antiatheraogenic effect was markedly manifested by a reduction of lipid plaques in the arteries in hypercholesterolemic animals, decreased accumulation of cholesterol in vascular walls, and other positive interventions.

[Pharmaceutical importance of Allium sativum L. 1. Organic sulfur compounds and their transformation based on present knowledge]. / Farmaceutický význam Allium sativum L. 1. Organické slouceniny síry a jejich transformace podle soucasných poznatku.

The paper sums up new experimental knowledge concerning the individual groups of organic sulfurous substances of the garlic: sulfoxides, thiosulfinate, ajoens, vinyldithiines, alkyl and alkene sulfides and glutamylpeptides of sulfurous amino acids, their transformation reactions (based on the temperature, pH, extraction medium, and time) and the final products of transformations (Scheme 1, 2). It deals with the activity of the enzyme alliinase necessary for the transformation of sulfoxides present in the whole garlic, its isolation and stability as well as the stability of the dominant thiosulfinate allicin in various media and simulated body fluids. It refers to the studies of the metabolism and transformations of the most important sulfurous components performed in vitro on the hepatocytes and on the isolated rat liver, and those carried out in vivo on the rats and including the examination of the composition of the exhaled air. It follows from published papers that all different degradation products of thiosulfinates, mainly the prevailing allicin, are carriers of various biological activities. The paper also lists the types of commercial preparations prepared from the garlic, their differences, and considerable variability of their contents of active principles.

[Pharmaceutical importance of Allium sativum L. 2. Antibacterial effects]. / Farmaceutický význam Allium sativum L. 2. Antibakteriální úcinek.

The communication summarizes mainly newly obtained experimental findings confirming the antibacterial effect of garlic preparations (powders, extracts, juice, essential oil, oil macerate) and their individual components. It also reports the effectiveness of substances newly isolated from the oil macerate (iso-E-10-devinylajoene, Z-10-devinylajoene, and three, or five thiosulfinates). The effect on the tested bacteria included here is, according to new evidence, indisputable. The paper has purposefully excluded the action of garlic against the widely distributed bacteria Helicobacter pylori, which causes chronic gastritis, gastric and duodenal ulcers. The findings concerning this matter will be published in the following communication.

[Pharmaceutical importance of Allium sativum L. 3. Antibacterial effects on Helicobacter pylori]. / Farmaceutický význam Allium sativum L. 3. Antibakteriální úcinek proti Helicobacter pylori.

The paper points out the risk factors which render possible the outbreak of infections due to the bacterium Helicobacter pylori manifesting itself as chronic gastritis. In a great extent it results in peptic and duodenal ulcers and can even lead to the development of adenocarcinoma and lymphoma of the stomach. The paper mentions the efficacy of previous and contemporary therapy. Possible use of garlic in the treatment of these infections is intensively investigated. At present mainly in vitro experiments showing promising results are performed. A minimum of experiments carried out with out-patients produced negative results. As they do not fulfill the parameters of clinical experiments, this question still remains open.

Higher anti-tumour efficacy of platinum(IV) complex LA-12 is associated with its ability to bypass M-phase entry block induced in oxaliplatin-treated human colon cancer cells.

OBJECTIVES: Therapeutic potential of conventionally used platinum-based drugs in treatment of colorectal tumours has been limited due to high incidence of tumour resistance to them and to their severe side effects. This evokes a search for more suitable anti-cancer drugs. We have compared ability of oxaliplatin and a novel platinum(IV) complex, LA-12, to modulate the cell cycle and induce apoptosis in human colon adenocarcinoma HCT116 wt and p53/p21 null cells, and have investigated molecular mechanisms involved. MATERIALS AND METHODS: Cell cycle-related changes were analysed by flow cytometry (bromodeoxyuridine/propidium iodide staining, histone H3 phosphorylation). Apoptosis was detected using flow cytometry (assays monitoring caspase activity) and fluorescence microscopy (nuclear morphology). Changes in levels of genes/proteins involved in cell cycle and apoptosis regulation were examined by RT-PCR and western blotting. RESULTS: Our results highlight the outstanding ability of LA-12 to induce effective elimination of colon cancer cells independently of p53/p21, and in significantly lower doses compared to oxaliplatin. While oxaliplatin induced p53- and p21-dependent G2 -phase arrest associated with downregulation of cyclin B1 and Cdk1, LA-12 allowed cells to enter M-phase of the cell cycle regardless of p53/p21 status. CONCLUSIONS: Higher malignant cell toxicity and ability to bypass cell cycle arrest important for the cell damage repair suggest LA-12 to be a more effective candidate for elimination of colon tumours from a variety of genetic backgrounds, compared with oxaliplatin.

Efficiency of viral DNA synthesis during infection of permissive and nonpermissive cells with vif-negative human immunodeficiency virus type 1.

The rate-limiting steps in infection by human immunodeficiency virus type 1 (HIV-1) deficient in the viral infectivity factor, Vif, are unknown. As a measurement of completion of the early stages of the HIV-1 life cycle, the levels of viral DNA were examined by polymerase chain reaction amplification during infection by vif-positive and vif-negative viruses of MT-2 and H9 cells, in which vif is required for HIV-1 replication. Viral DNA was detected within hours of infection by both viruses, but the accumulation of vif-negative virus DNA was impeded in terms of both extent and kinetics. Inefficient viral DNA synthesis correlated with restricted replication of the vif-negative virus. Increasing the input dose of vif-negative virus increased viral DNA levels within 24 h of infection but failed to overcome the block to subsequent DNA synthesis and productive infection. Infection of C8166 cells, in which vif function is dispensable, resulted in efficient DNA synthesis by vif-positive and vif-negative viruses. We conclude that one defect in the replication of vif-negative HIV-1 in nonpermissive cells occurs prior to or during viral DNA synthesis and may reflect processes required for efficient nucleocapsid internalization or activation of reverse transcription.

The redox state of cysteines in human immunodeficiency virus type 1 Vif in infected cells and in virions.

HIV-1 Vif has two conserved cysteine residues in positions 114 and 133, both of which were found to be essential for HIV-1 infection and for Vif function in transcomplementation assays (X-Y. Ma, P. Sova, W. Chao, and D. J. Volsky, 1994, J. Virol. 68: 1714-1720). We evaluated here the redox status and disulfide bond formation of Vif cysteines inside cells or in virions and tested the role of Vif cysteines in Vif distribution in cells and in virions. Immunoblot analysis of Vif in wild type virus-infected cells and virions under different redox conditions revealed that the cysteine residues are readily accessible to chemical interaction but they do not form intramolecular disulfide bonds either inside cells or in virions, nor do they form covalent bonds with other proteins in either compartment. Cysteine mutants of Vif resembled wildtype Vif in their intracellular and virion distribution, indicating that Vif cysteines do not affect intracellular Vif transport and packaging into virions. We conclude that the cysteines in Vif do not form sulfhydryl bonds either intracellularly or in virions and may contribute to Vif activity rather than structure.

Vif is largely absent from human immunodeficiency virus type 1 mature virions and associates mainly with viral particles containing unprocessed gag.

Vif is a human immunodeficiency virus type 1 (HIV-1) protein that is essential for the production of infectious virus. Most of Vif synthesized during HIV infection localizes within cells, and the extent of Vif packaging into virions and its function there remain controversial. Here we show that a small but detectable amount of Vif remains associated with purified virions even after their treatment with the protease subtilisin. However, treatment of these virions with 1% Triton X-100 revealed that most of the virion-associated Vif segregated with detergent-resistant virus particles consisting of unprocessed Gag, indicating that detergent-soluble, mature virions contain very little Vif. To investigate the control of Vif packaging in immature virus particles, we tested its association with Gag-containing virus-like particles (VLPs) in a Vif and Gag coexpression system in human cells. Only a small proportion of Vif molecules synthesized in this system became packaged into VLPs, and the VLP-associated Vif was protected from exogenous protease and detergent treatment, indicating that it is stably incorporated into immature virion-like cores. About 10-fold more Vpr than Vif was packaged into VLPs but most of the VLP-associated Vpr was removed by treatment with detergent. Mutagenesis of the C-terminal sequences in Gag previously shown to be responsible for interaction with Vif did not reduce the extent of Vif packaging into Gag VLPs. Surprisingly, short deletions in the capsid domain (CA) of Gag (amino acid residues 284 to 304 and 350 to 362) increased Vif packaging over 10-fold. The 350 to 363 deletion introduced into CA in HIV provirus also increased Vif incorporation into purified virions. Our results show that Vif can be packaged at low levels into aberrant virus particles or immature virions and that Vif is not present significantly in mature virions. Overall, these results indicate that the Vif content in virions is tightly regulated and also argue against a function of virion-associated Vif.

Cysteine residues in the Vif protein of human immunodeficiency virus type 1 are essential for viral infectivity.

The infectivity factor of human immunodeficiency virus type 1 (HIV-1), Vif, contains two cysteine residues which are highly conserved among animal lentiviruses. We introduced substitutions of leucine for cysteine residues in the vif gene of a full-length HIV-1 clone to analyze their roles in viral infection. Mutant viruses containing substitutions in either Cys-114, Cys-133, or both displayed a vif-negative infection phenotype similar to that of an isogeneic vif deletion mutant, namely, a cell-dependent complete to partial loss of infectivity. The vif defect could be complemented by cotransfection of mutant viral DNA with a Vif expression vector, and there was no evidence that recombination contributed to the repair of the vif deficiency. The viral protein profile, as determined by immunoblotting, in cells infected with cysteine substitution mutants and that in wild-type virus were similar, including the presence of the 23-kDa Vif polypeptide. In addition, immunoblotting with an antiserum directed against the carboxyl terminus of gp41 revealed that gp41 was intact in cells infected with either wild-type or vif mutant HIV-1, excluding that Vif cleaves the C terminus of gp41. Our results indicate that the cysteines in HIV-1 Vif are critical for Vif function in viral infectivity.

Peptide inhibitors of HIV-1 protease and viral infection of peripheral blood lymphocytes based on HIV-1 Vif.

We recently reported that HIV-1 Vif (virion infectivity factor) inhibits HIV-1 protease in vitro and in bacteria, suggesting that it may serve as the basis for the design of new protease inhibitors and treatment for HIV-1 infection. To evaluate this possibility, we synthesized peptide derivatives from the region of Vif, which inhibits protease, and tested their activity on protease. In an assay of cleavage of virion-like particles composed of HIV-1 Gag precursor polyprotein, full-length recombinant Vif, and a peptide consisting of residues 21-65 of Vif, but not a control peptide or BSA, inhibited protease activity. Vif21-65 blocked protease at a molar ratio of two to one. We then tested this peptide and a smaller peptide, Vif41-65, for their effects on HIV-1 infection of peripheral blood lymphocytes. Both Vif peptides inhibited virus expression below the limit of detection, but control peptides had no effect. To investigate its site of action, Vif21-65 was tested for its effect on Gag cleavage by protease during HIV-1 infection. We found that commensurate with its reduction of virus expression, Vif21-65 inhibited the cleavage of the polyprotein p55 to mature p24. These results are similar to those obtained by using Ro 31-8959, a protease inhibitor in clinical use. We conclude that Vif-derived peptides inhibit protease during HIV-1 infection and may be useful for the development of new protease inhibitors.

Mapping of serologically relevant regions of human cytomegalovirus phosphoprotein pp150 using synthetic peptides.

The entire amino acid sequence of human cytomegalo-virus (HCMV) 150K matrix phosphoprotein (pp150), consisting of 1048 amino acid residues, was divided into 95 overlapping 20 amino acid peptides which were synthesized on polyethylene rods. The rods were subjected to ELISA with pooled anti-HCMV-positive and anti-HCMV-negative sera. Four peptides recognized by the anti-HCMV-positive pool only were synthesized by the solid-phase method and their reactivity in a conventional ELISA, using a panel of 14 individual anti-HCMV-negative and 20 anti-HCMV-positive antisera, was evaluated; three peptides were found to be specifically reactive. Results obtained with one of these peptides (residues 595 to 614) in ELISA showed a good correlation with those obtained using a routinely performed complement fixation test.

Endothelial cell growth factors in embryonic and adult chick brain are related to human acidic fibroblast growth factor.

We have investigated the nature of endothelial cell growth factors in 14-day embryonic and adult chick brain extracts. Mitogenic activity was isolated by a combination of cation-exchange, heparin-Sepharose affinity, and reverse-phase HPLC. Two major mitogenic fractions eluted from heparin-Sepharose at 0.8-1.3 M and 1.5-2 M. Biologically active proteins eluting at 0.8-1.3 M NaCl, after purification to homogeneity from embryonic and adult brain, were found to possess the same amino-terminal sequence as human acidic fibroblast growth factor (aFGF). The notion that the isolated mitogens represent chick aFGF is further supported by the findings that their affinity for heparin and their retention behavior in highly resolutive HPLC are indistinguishable from those of genuine aFGF. Mitogenic activities eluting at 1.5-2 M NaCl were also present in embryonic and adult brain, but in quantities insufficient for preliminary characterization. The high specific mitogenic activity for endothelial cells, high affinity for heparin and cross-reactivity with antibodies against bovine basic FGF (bFGF) suggest a relationship of those materials with basic FGF. Our data also suggest that the sequence of aFGF is highly conserved among vertebrates. While angiogenesis occurs predominantly in the embryonic brain, the absence of notable differences in the contents of the potent angiogenic factors aFGF and bFGF in embryonic versus adult chick brain is interesting.

Amino acid sequence of human acidic fibroblast growth factor.

The complete amino acid sequence of human brain acidic fibroblast growth factor (aFGF) has been established. Human aFGF consists of 140 amino acids and is highly homologous to bovine aFGF (11 amino acid replacements). Results from experiments involving alkylation of cysteine residues are compatible with the possibilities that in aFGF all three cysteines exist as free sulfhydryls, or alternatively, that a disulfide bridge is present but cannot be identified due to disulfide scrambling caused by the SH group of the remaining cysteine. A potential glycosylation site Asn114-Gly115-Ser116 is present in aFGF but the mitogen does not bind to lectins suggesting that it may not be glycosylated.